Theses and Dissertations
This collection includes both ASU Theses and Dissertations, submitted by graduate students, and the Barrett, Honors College theses submitted by undergraduate students.
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- All Subjects: Biophysics
- Genre: Doctoral Dissertation
Description
Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only a hundred unit cells with sizes of less than 200 nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet would bring fully hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses ( 70 fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. At the initial experiments at the AMO beamline using 6.9- Å wavelength, Bragg peaks were recorded to 8.5- Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage. Recently, femtosecond X-ray protein nanocrystallography experiments were done at the CXI beamline of the LCLS using 1.3- Å wavelength, and Bragg reflections were recorded to 3- Å resolution; the data are currently being processed. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.
ContributorsHunter, Mark (Author) / Fromme, Petra (Thesis advisor) / Wolf, George (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2011
Description
Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the ERK2 protein kinase. I performed molecular dynamics simulations of the lac repressor headpiece - O1 operator complex at the natural angle as well as at under- and overbent angles to assess the factors that determine the natural DNA bending angle. I find both energetic and entropic factors contribute to recognition of the natural angle. At the natural angle the energy of the system is minimized by optimization of protein-DNA contacts and the entropy of the system is maximized by release of water from the protein-DNA interface and decorrelation of protein motions. To identify the mechanism by which mutations lead to auto-activation of ERK2, I performed a series of molecular dynamics simulations of ERK1/2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P and R65S ERK2 mutants. My simulations indicate the importance of domain closure for auto-activation and activity regulation. My results enable me to predict two loss-of-function mutants of ERK2, G83A and Q64C, that have been confirmed in experiments by collaborators. One of the powerful capabilities of MD simulations in biochemistry is the ability to find low free energy pathways that connect and explain disparate structural data on biomolecular systems. An extention of the targeted molecular dynamics technique using constraints on internal coordinates will be presented and evaluated. The method gives good results for the alanine dipeptide, but breaks down when applied to study conformational changes in GroEL and adenylate kinase.
ContributorsBarr, Daniel Alan (Author) / van der Vaart, Arjan (Thesis advisor) / Matyushov, Dmitry (Committee member) / Wolf, George (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
This thesis describes several approaches to next generation DNA sequencing via tunneling current method based on a Scanning Tunneling Microscope system. In chapters 5 and 6, preliminary results have shown that DNA bases could be identified by their characteristic tunneling signals. Measurements taken in aqueous buffered solution showed that single base resolution could be achieved with economic setups. In chapter 7, it is illustrated that some ongoing measurements are indicating the sequence readout by making linear scan on a piece of short DNA oligomer. However, to overcome the difficulties of controlling DNA especially ssDNA movement, it is much better to have the tunneling measurement incorporated onto a robust nanopore device to realize sequential reading of the DNA sequence while it is being translocated.
ContributorsHuang, Shuo (Author) / Lindsay, Stuart (Thesis advisor) / Sankey, Otto (Committee member) / Tao, Nongjian (Committee member) / Drucker, Jeff (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2011
Description
This thesis describes several experiments based on carbon nanotube nanofludic devices and field-effect transistors. The first experiment detected ion and molecule translocation through one single-walled carbon nanotube (SWCNT) that spans a barrier between two fluid reservoirs. The electrical ionic current is measured. Translocation of small single stranded DNA oligomers is marked by large transient increases in current through the tube and confirmed by a PCR (polymerase chain reaction) analysis. Carbon nanotubes simplify the construction of nanopores, permit new types of electrical measurement, and open new avenues for control of DNA translocation. The second experiment constructed devices in which the interior of a single-walled carbon nanotube field-effect transistor (CNT-FET) acts as a nanofluidic channel that connects two fluid reservoirs, permitting measurement of the electronic properties of the SWCNT as it is wetted by an analyte. Wetting of the inside of the SWCNT by water turns the transistor on, while wetting of the outside has little effect. This finding may provide a new method to investigate water behavior at nanoscale. This also opens a new avenue for building sensors in which the SWCNT functions as an electronic detector. This thesis also presents some experiments that related to nanofabrication, such as construction of FET with tin sulfide (SnS) quantum ribbon. This work demonstrates the application of solution processed IV-VI semiconductor nanostructures in nanoscale devices.
ContributorsCao, Zhai (Author) / Lindsay, Stuart (Thesis advisor) / Vaiana, Sara (Committee member) / Ros, Robert (Committee member) / Marzke, Robert (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Biological membranes are critical to cell sustainability by selectively permeating polar molecules into the intracellular space and providing protection to the interior organelles. Biomimetic membranes (model cell membranes) are often used to fundamentally study the lipid bilayer backbone structure of the biological membrane. Lipid bilayer membranes are often supported using inorganic materials in an effort to improve membrane stability and for application to novel biosensing platforms. Published literature has shown that a variety of dense inorganic materials with various surface properties have been investigated for the study of biomimetic membranes. However, literature does not adequately address the effect of porous materials or supports with varying macroscopic geometries on lipid bilayer membrane behavior. The objective of this dissertation is to present a fundamental study on the synthesis of lipid bilayer membranes supported by novel inorganic supports in an effort to expand the number of available supports for biosensing technology. There are two fundamental areas covered including: (1) synthesis of lipid bilayer membranes on porous inorganic materials and (2) synthesis and characterization of cylindrically supported lipid bilayer membranes. The lipid bilayer membrane formation behavior on various porous supports was studied via direct mass adsorption using a quartz crystal microbalance. Experimental results demonstrate significantly different membrane formation behaviors on the porous inorganic supports. A lipid bilayer membrane structure was formed only on SiO2 based surfaces (dense SiO2 and silicalite, basic conditions) and gamma-alumina (acidic conditions). Vesicle monolayer adsorption was observed on gamma-alumina (basic conditions), and yttria stabilized zirconia (YSZ) of varying roughness. Parameters such as buffer pH, surface chemistry and surface roughness were found to have a significant impact on the vesicle adsorption kinetics. Experimental and modeling work was conducted to study formation and characterization of cylindrically supported lipid bilayer membranes. A novel sensing technique (long-period fiber grating refractometry) was utilized to measure the formation mechanism of lipid bilayer membranes on an optical fiber. It was found that the membrane formation kinetics on the fiber was similar to its planar SiO2 counterpart. Fluorescence measurements verified membrane transport behavior and found that characterization artifacts affected the measured transport behavior.
ContributorsEggen, Carrie (Author) / Lin, Jerry Y.S. (Thesis advisor) / Dai, Lenore (Committee member) / Rege, Kaushal (Committee member) / Thornton, Trevor (Committee member) / Vogt, Bryan (Committee member) / Arizona State University (Publisher)
Created2011
Description
ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure.
ContributorsLawrence, Robert Michael (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian J.L. (Committee member) / Woodbury, Neal W. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Nanofluidic devices in which one single-walled carbon nanotube (SWCNT) spans a barrier between two fluid reservoirs were constructed, enabling direct electrical measurement of the transport of ions and molecules. Ion current through these devices is about 2 orders of magnitude larger than that predicted from the bulk resistivity of the electrolyte. Electroosmosis drives excess current, carried by cations, and is found to be the origin of giant ionic current through SWCNT as shown by building an ionic field-effect transistor with a gate electrode embedded in the fluid barrier. Wetting of inside of the semi-conducting SWCNT by water showed the change of its electronic property, turning the electronic SWCNT field-effect transistor to "on" state. These findings provide a new method to investigate and control the ion and molecule behavior at nanoscale.
ContributorsPang, Pei (Author) / Lindsay, Stuart (Thesis advisor) / Ros, Robert (Committee member) / Shumway, John (Committee member) / Tao, Nongjian (Committee member) / Menéndez, Jose (Committee member) / Arizona State University (Publisher)
Created2011
Description
Proteins are a fundamental unit in biology. Although proteins have been extensively studied, there is still much to investigate. The mechanism by which proteins fold into their native state, how evolution shapes structural dynamics, and the dynamic mechanisms of many diseases are not well understood. In this thesis, protein folding is explored using a multi-scale modeling method including (i) geometric constraint based simulations that efficiently search for native like topologies and (ii) reservoir replica exchange molecular dynamics, which identify the low free energy structures and refines these structures toward the native conformation. A test set of eight proteins and three ancestral steroid receptor proteins are folded to 2.7Å all-atom RMSD from their experimental crystal structures. Protein evolution and disease associated mutations (DAMs) are most commonly studied by in silico multiple sequence alignment methods. Here, however, the structural dynamics are incorporated to give insight into the evolution of three ancestral proteins and the mechanism of several diseases in human ferritin protein. The differences in conformational dynamics of these evolutionary related, functionally diverged ancestral steroid receptor proteins are investigated by obtaining the most collective motion through essential dynamics. Strikingly, this analysis shows that evolutionary diverged proteins of the same family do not share the same dynamic subspace. Rather, those sharing the same function are simultaneously clustered together and distant from those functionally diverged homologs. This dynamics analysis also identifies 77% of mutations (functional and permissive) necessary to evolve new function. In silico methods for prediction of DAMs rely on differences in evolution rate due to purifying selection and therefore the accuracy of DAM prediction decreases at fast and slow evolvable sites. Here, we investigate structural dynamics through computing the contribution of each residue to the biologically relevant fluctuations and from this define a metric: the dynamic stability index (DSI). Using DSI we study the mechanism for three diseases observed in the human ferritin protein. The T30I and R40G DAMs show a loss of dynamic stability at the C-terminus helix and nearby regulatory loop, agreeing with experimental results implicating the same regulatory loop as a cause in cataracts syndrome.
ContributorsGlembo, Tyler J (Author) / Ozkan, Sefika B (Thesis advisor) / Thorpe, Michael F (Committee member) / Ros, Robert (Committee member) / Kumar, Sudhir (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
The properties of materials depend heavily on the spatial distribution and connectivity of their constituent parts. This applies equally to materials such as diamond and glasses as it does to biomolecules that are the product of billions of years of evolution. In science, insight is often gained through simple models with characteristics that are the result of the few features that have purposely been retained. Common to all research within in this thesis is the use of network-based models to describe the properties of materials. This work begins with the description of a technique for decoupling boundary effects from intrinsic properties of nanomaterials that maps the atomic distribution of nanomaterials of diverse shape and size but common atomic geometry onto a universal curve. This is followed by an investigation of correlated density fluctuations in the large length scale limit in amorphous materials through the analysis of large continuous random network models. The difficulty of estimating this limit from finite models is overcome by the development of a technique that uses the variance in the number of atoms in finite subregions to perform the extrapolation to large length scales. The technique is applied to models of amorphous silicon and vitreous silica and compared with results from recent experiments. The latter part this work applies network-based models to biological systems. The first application models force-induced protein unfolding as crack propagation on a constraint network consisting of interactions such as hydrogen bonds that cross-link and stabilize a folded polypeptide chain. Unfolding pathways generated by the model are compared with molecular dynamics simulation and experiment for a diverse set of proteins, demonstrating that the model is able to capture not only native state behavior but also partially unfolded intermediates far from the native state. This study concludes with the extension of the latter model in the development of an efficient algorithm for predicting protein structure through the flexible fitting of atomic models to low-resolution cryo-electron microscopy data. By optimizing the fit to synthetic data through directed sampling and context-dependent constraint removal, predictions are made with accuracies within the expected variability of the native state.
ContributorsDe Graff, Adam (Author) / Thorpe, Michael F. (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Matyushov, Dmitry (Committee member) / Ozkan, Sefika B. (Committee member) / Treacy, Michael M. J. (Committee member) / Arizona State University (Publisher)
Created2011
Description
Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed.
ContributorsSpiriti, Justin Matthew (Author) / van der Vaart, Arjan (Thesis advisor) / Chizmeshya, Andrew (Thesis advisor) / Matyushov, Dmitry (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011